Approximately 160 million American men, women and children are overweight or obese. Almost 75% of men, 60% of women and 30% of boys and girls under the age of 20 are obese or overweight. Obesity is associated with numerous co-morbidities including diabetes, cardiovascular disease, and cancers. There are two types of fat. Brown fat is common in newborns and small mammals. It is useful for generating heat, especially in small bodies that tend to lose heat. White fat increases as people age and is useful for storing energy and insulating against heat loss. Increased white fat is associated with increased obesity-related disease whereas increased brown fat is associated with reduced obesity-related disease. Several recent studies of adipose tissue have identified transcriptional signatures associated with brown fat adipogenesis and specifically thermogenic programs. Some of these studies have also identified long non- coding RNAs (lncRNAs) associated with white fat and brown fat identity and function. However, the mechanism(s) of how these lncRNAs affect transcriptional programs and determine brown fat identity and function are poorly understood. We propose to use our recently developed lncRNA-based yeast three hybrid (Y3H) assay to systematically define lncRNA-protein interactions for 318 conserved lncRNAs implicated in brown fat adipogenesis. Our Y3H system is capable of detecting lncRNA-protein interactions that are otherwise difficult to detect due to issues of low-abundance and low-affinity interactions. Overlaying lncRNA-protein interactions onto existing protein- protein interaction networks will define lncRNA-regulated pathways of normal brown fat adipogenesis and will also identify lncRNA-regulated pathways that might be manipulated to increase thermogenesis. Because we will screen overlapping lncRNA fragments of these 318 individual lncRNAs, these studies will enable systematic lncRNA structure-function analyses. The information obtained through this structure-function analysis will be critical for establishing sequence and/or structural parameters which may enable prediction of lncRNA-protein binding activity, thereby advancing our understanding of the biology of lncRNAs, even those not included in this initial screen. For a set of lncRNA-protein interactions, we will validate roles in mouse models of brown fat adipogenesis by increasing (by ectopic expression) or decreasing (by knockdown) lncRNAs and their associated proteins in brown fat progenitor cells. We will assess the effects of these interactions by testing obesity, brown fat lineage commitment (by FACS analysis) and thermogenesis (by transcriptional profiling). Thus, these studies will (1) identify lncRNA-protein interactions directing adipogenesis and thermogenesis; (2) develop a platform for systemically studying lncRNAs; and (3) provide pre-clinical evidence support clinical trials targeting lncRNA- directed pathways for obesity-related diseases.
PROJECT NARATIVE These studies will identify long non-coding RNAs (lncRNAs) and their interacting proteins important for brown fat adipogenesis and function. These studies will also identify specific lncRNA-protein interactions that when disrupted could remediate obesity-related disease.
